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  • C07C59/235—Saturated compounds containing more than one carboxyl group
  • C07C59/305—Saturated compounds containing more than one carboxyl group containing ether groups, groups, groups, or groups
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  • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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  • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
• • A—HUMAN NECESSITIES
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/465—Streptomyces
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
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  • C12R2001/745—Cephalosporium ; Acremonium
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
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  • C12R2001/80—Penicillium
  • C12R2001/82—Penicillium chrysogenum
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to antibiotics and in particular to novel beta lactam antibiotics and their production.
• beta lactams constitute a group of antibiotics of particular interest into which considerable research has taken place with the aim of producing new antibiotic compounds.
• the biosynthesis of isopenicillin N from D-valine, L-cysteine and L- ⁇ -amioadipic acid proceeds via the tripeptide ⁇ -(L- ⁇ -aminoadipyl-L-cysteinyl-D-valine (throughout this specification "adipyl” is used to denote the monovalent radical derived from adipic acid) through the action on this tripeptide of the enzyme isopenicillin N synthetase.
• the present invention comprises a compound of the formula ##STR2## wherein X is sulphur or methylene, R 1 is hydrogen, amino or an acylated or carbamylated amino group, R 2 is hydrogen or an alkyl group and R 3 is an alkoxy group.
• Such penicillin compounds (I) are of course very unusual in that they contain an alkoxy group and either hydrogen or an alkyl group at the 2-position of the penam system instead of the usual two alkyl (methyl) groups.
• X is preferably a methylene group rather than sulphur.
• R 1 is amino or an acylated or carbamylated amino group are of particular interest.
• Substituted amino groups preferably have the form RCONH-- or RNHCONH-- where R is hydrogen or a lower (C 1 to C 5 and preferably C 1 to C 3 ) alkyl group, or alternatively a phenyl or substituted phenyl group.
• Such substituted phenyl groups may contain one or more of various types of substituent including alkyl and alkoxy groups, for example those being or containing alkyl groups of 1, 2 or 3 or more carbon atoms as described below for R 2 and especially methyl and methoxy, substituents being or containing a halogen residue, for example bromo, chloro and fluoro and halogen substituted alkyl groups such as CF 3 , free and substituted amino groups, for example dimethylamino, hydroxy and nitro groups, as well as other benzene ring substituting groups known in the art.
• substituents being or containing a halogen residue, for example bromo, chloro and fluoro and halogen substituted alkyl groups such as CF 3
• free and substituted amino groups for example dimethylamino, hydroxy and nitro groups, as well as other benzene ring substituting groups known in the art.
• compounds containing an unsubstituted amino group R 1 are of greater interest than
• R 2 this is preferably an alkyl group and R 2 and also R 3 may conveniently each be separately selected from groups of 1 to 5 carbon atoms, especially of 1 to 3 or 4 carbon atoms.
• groups conveniently are or contain a straight chain alkyl group, the smaller groups such as isopropyl, propyl, particularly ethyl and especially methyl, and isopropoxy, propoxy, particularly ethoxy and especially methoxy, being most preferred.
• the penicillins (I) may be in the form of a physiologically aceeptable salt thereof.
• Such salt formation may involve the amino group and a physiologically acceptable inorganic or organic acid or one or both of the carboxyl groups and a physiologically acceptable inorganic or organic base, the sodium and potassium salts of the penicillin compounds (I), for example, being of some interest.
• R 1 is other than hydrogen
• this carbon atom is of the L configuration.
• R 2 and R 3 there are two possible configurations at the asymmetric carbon atom to which these groups are attached, depending on whether the group R 2 or the group R 3 is in a similar spatial dispostion to the adjacent carboxyl group.
• the latter alternative is of particular interest, especially in the case where R 2 is alkyl, since such an isomer is that directly generated by the process described herein, as discussed hereinafter in more detail.
• Specific pencillins according to the present invention are the compounds (I) in which X is sulphur or methylene, R 1 is hydrogen or amino, R 2 is hydrogen, methyl or ethyl, and R 3 is methoxy or ethoxy, including particularly those compounds in which R 2 is hydrogen or methyl and R 3 is methoxy, such as the compounds 2-methoxy-2-methyl-6- ⁇ -(L- ⁇ -aminoadipamido)-3-carboxypenam, 2-methoxy-2-methyl-6- ⁇ -(D- ⁇ -aminoadipamido)-3-carboxypenam, 2-methoxy-2-methyl-6- ⁇ -adipamido-3-carboxypenam and 2-methoxy-2-methyl-6- ⁇ -(L-S-carboxymethylcysteinyl)-3-carboxypenam, 2-methoxy-6- ⁇ -(L- ⁇ -aminoadipamido)-3-carboxypenam, 2-methoxy-6- ⁇ -(D-
• penicillins (I) are of particular interest, especially where they contain a group R 2 which is alkyl, as the stereoisomer in which the alkoxy and carboxy groups at the 2- and 3-positions of the thiazolidone ring have a similar spatial disposition, being in the cis configuration, i.e. as the alkoxy 2S-isomer.
• R 2 which is alkyl
• the structure of the compound 2S-2-methoxy-2-methyl-6- ⁇ -(L- ⁇ -aminoadipamido)-3-carboxypenam is illustrated below in formula (II).
• formula (II) the adjacent amino and carboxy groups are shown in the zwitterion form in which they usually exist. ##STR3##
• the penicillins (I) in which R 1 is hydrogen or amino may conveniently be prepared from the corresponding tripeptide of formula ##STR4## wherein X is sulphur or methylene, R 1 is hydrogen or amino, R 2 is hydrogen or an alkyl group and R 3 is an alkoxy group, through the action of the enzyme isopenicillin N synthetase.
• X is sulphur or methylene
• R 1 is hydrogen or amino
• R 2 is hydrogen or an alkyl group
• R 3 is an alkoxy group
• the penicillin (I) can, as explained hereinbefore, exist in two stereoisomeric forms differing in the disposition of the groups at the C 2 position of the thiazolidone ring.
• the tripeptide (III) will similarly contain an asymmetric carbon atom at the equivalent position and it has been found that efficient conversion of the tripeptide (III) to the penicillin (I) with isopenicillin N synthetase occurs only with the tripeptide having the R-configuration at the 2-position, this configuration being retained during the conversion to give the stereochemically equivalent penicillin having the S-configuration at the 2-position.
• R 2 is hydrogen, the carbon atom at the equivalent position in the tripeptide is not asymmetric and it is believed that the penicillin obtained is a mixture of the two stereoisomeric forms differing at the C 2 position.
• tripeptides (III) used to prepare the penicillins (I) are novel compounds and are included within the scope of the present invention in their monomeric or disulphide form (tripeptides in the latter form are converted to the monomeric form by the use of dithiothreitol as described hereinafter) as are derivatives thereof in which one or more functional groups therein are in protected form.
• the sulphydryl group of the central cysteinyl residue, the carboxy group of the C-terminal residue, and the carboxy and amino (where present) groups of the N-terminal residue may be protected, for example by conventional protecting groups used in peptide chemistry and as illustrated in the Examples, such protected tripeptides being used in the synthesis of the final, unprotected tripeptide (III).
• tripeptides are compounds (III) in which X is sulphur or methylene, R 1 is hydrogen or amino, R 2 is hydrogen, methyl or ethyl, and R 3 is methoxy or ethoxy, including particularly those compounds in which R 2 is hydrogen or methyl and R 3 is methoxy, such as the compounds ⁇ -(L- ⁇ -aminoadipyl)-L-cysteinyl-(2R-2-amino-3-methoxybutanoic acid), ⁇ -(D- ⁇ -aminoadipyl)-L-cysteinyl-(2R-2-amino-3-methoxybutanoic acid), ⁇ -adipyl-L-cysteinyl-(2R-2-amino-3-methoxybutanoic acid), ⁇ -(L-S-carboxymethylcysteinyl)-L-cysteinyl-(2R-2-amino-3-methoxybutanoic acid), ⁇ -
• the tripeptides (III) are readily synthesised by standard procedures of peptide chemistry.
• a suitable route generally involves the coupling of protected forms of the individual amino acids constituting the tripeptide and subsequent deprotection.
• Suitable protecting groups may be selected from those routinely used in the art of peptide synthesis, for example amino groups being protected as benzyloxycarbonyl or like derivatives, carboxy groups as benzyl or other esters and sulphydryl groups as the benzyl derivative.
• the tripeptides (III) are subjected to the action of isopenicillin N synthetase.
• This enzyme is produced by various micro-organisms, for example Penicillin chrysogenum and Streptomyces clavuligenis but a preferred source is Cephalosporium acremonium.
• the micro-organism C. acremonium is widely available so that, apart from other sources, several strains are available from the American Type Culture Collection, for example ATCC 20339 (Cephalosporium sp. strain F.12), ATCC 14553 (C. acremonium) and ATCC 36255 (Acremonium strictum).
• the cells are cultured under suitable conditions and a cell free extract then procuded which may be purified by a combination of procedures known in the art for the purification of proteins.
• a suitable purification procedure is illustrated in the Examples and this will provide an enzyme suitable for use in conversion of the tripeptide to an antibiotic compound, generally providing a level of conversion of about 20% or higher for the tripeptides containing an alkyl group R 2 .
• An enzyme of a level of purity such as it obtained by this procedure is free from activity which would extend the conversion beyond the isopenicillin stage.
• Incubation of the compound with the enzyme in an aqueous medium at a pH in the range from 7.0 to 8.0, for example 7.5, at a temperature in the range from 25°-28° C., for example 27° C., and for a period of 30 to 60 minutes, for example 30 minutes, is generally suitable, following which the antibiotic compound is isolated by the use of conventional procedures.
• one or more cofactors in particular one or more, and conveniently all three, of iron, especially ferrous ions as provided for example by ferrous sulphate, ascorbate ions as provided for example by ascorbic acid, and dithiothreitol.
• Catalase may also be incorporated with advantage into the incubation medium. A procedure using these added components is illustrated in the Examples.
• Penicillins (I) containing an acylated or carbamylated amino group may conveniently be prepared from the corresponding penicillin (I) containing an amino group by the action of a suitable acylating agent, for example using the appropriate acid or a derivative thereof, such ad the chloride or anhydride, or of a suitable carbamylating agent, such as an appropriate alkali metal cyanate. Salts may conveniently be prepared using the appropriate inorganic or organic acid or base.
• Penicillin compounds according to the present invention particularly the penicillin 2S-2-methoxy-2-methyl-6- ⁇ -(L- ⁇ -aminoadipamido)-3-carboxypenam and 2-methoxy-6- ⁇ -(L- ⁇ -aminoadipamido)-3-carboxy penam have given very promising results in tests with Staphylococcus aureus and are also of interest for use in the control of a wide range of bacteria, both Gram-negative and Gram-positive, for example Escherichia coli, Salmonella typhimurium and Bacillus magaterium. Thus, a level of activity is observed which is at least comparable with that or isopenicillin N itself and it will be appreciated that these compounds therefor present a valuable alternative source of antibiotics.
• the compounds may be formulated for use as pharmaceuticals, for veterinary or particularly for human use, by a variety of methods but usually involving the use of a physiologically acceptable diluent or carrier.
• a physiologically acceptable diluent or carrier for instance, they may be applied as an aqueous, oily or emulsified composition incorporating a liquid diluent which most usually will be employed for parenteral administration and therefore will be sterile and pyrogen free.
• the compound may be formulated with a liquid diluent for oral administration, although it is more usually preferred to use for this purpose compositions incorporating a solid carrier, for example a conventional solid carrier material such as starch, lactose, dextrin or magnesium stearate.
• compositions may be formulated in unit dosage form, i.e. in the form of discrete portions each comprising a unit dose, or a multiple or sub-multiple of a unit does. Whilst the dosage of active compound given will depend on various factors, including the particular compound which is employed in the composition, it may be stated by way of guidance that levels broadly similar to those used for existing penicillins may be employed, i.e. daily dosage levels for adult humans being of the order of 250 mg to 5 g, often 500 g to 2 g, usually divided, although levels both above and below this may also be used in some cases; doses for children and for veterinary use will generally be adapted on a similar mg/kg basis. Where desired, more than one compound according to the present invention may be administered in the pharmaceutical composition or, indeed, other active compounds may be included in the composition.
• the invention thus includes a pharmaceutical composition comprising a compound (I) as described hereinbefore together with a physiologically acceptable diluent or carrier. Moreover, it also includes a method for the treatment of a patient suffering from a bacterial infection which comprises administering to that patient an amount of a compound (I) as described hereinbefore which is effective to counter that infection.
• Crotonic acid (8.6 g, 100 mmol) is dissolved in methanol (50 ml) and N-bromacetamide (13.8 g, 10 mmol) is added in portions over 30 minutes. The solution is stirred at 25° C. for 15 hours and the solvent is then evaporated and the residue partitioned between diethyl ether and water. The ether layer is dried (Na 2 SO 4 ), filtered, evaporated and the residue distilled to yield the title compounds as a colourless oil (14.3 g, 72%), b.p. 88°-89°/0.5 mm.
• 2R,3R- and 2S,3S-2-Bromo-3-methoxybutanoic acid (14 g, 71 mmol) are dissolved in ammonia (s.g. 0.88, 250 ml) and the mixture heated in an autoclave at 95° C. for 8 hours. The mixture is cooled to 25° C., evaporated, and the residue suspended in acetone. The resultant colourless solid is filtered off and washed with acetone to give the title compounds as a colourless solid containing residual ammonium bromide, (12.1 g) m.p. 180°-184° C. (dec.).
• 2R,3R- and 2S,3S-2-Amino-3-methoxybutanoic acid (3.1 g) are dissolved in a mixture of 1M sodium hydroxide (27 ml), water (20 ml), and dioxan (40 ml).
• Benzyl chloroformate (3.8 ml, 22 mmol) in dioxan (20 ml) and 1M sodium hydroxide (27 ml) are added to the solution individually, each at the same rate of addtion, over 25 minutes.
• N-p-Methoxybenzyloxycarbonyl-D-serine (5.39 mg, 2.00 mmol; prepared as a colourless solid of m.p. 96°-97° C. in 60% yield by the procedure of Weygand and Hunger, Berichte, 1962, 95 (1), 1) is dissolved in methanol and treated with a solution of potassium carbonate (138 mg, 1.00 mmol) in water (2 ml).
• N-p-methoxybenzyloxycarbonyl-D-serine benzyl ester (720 mg, 2.00 mmol) is dissolved in dry dichloromethane (20 ml) and the solution cooled to dry ice/acetone.
• Boron trifluoride etherate (100 ⁇ l) is added followed by the portionwise addition of diazomethane (30 mmol in 20 ml CH 2 Cl 2 ). After about 30 minutes the solution is filtered and washed once with water (10 ml), then dried (MgSO 4 ) and evaporated to dryness.
• N-p-methoxybenzyloxycarbonyl-O-methyl-D-serine benzyl ester 130 mg, 0.35 mmol
• TFA cold trifluoroacetic acid
• anisole 4.8:1 v/v
• the mixture is shaken on an ice until all the solid has dissolved and the TFA is then evaporated initially on a rotary evaporator at 0° C., finally using a high vacuum pump.
• ⁇ -(L- ⁇ -Aminoadipyl)-L-cysteinyl-(2R,3R,-2-amino-3-methoxybutanoic acid (210 ⁇ g: prepared as described in Example 1) is incubated with purified isopenicillin N synthetase (1.1 units, obtained as described below) in the presence of the cofactors dithiothreitol (2.11 mM), ascorbic acid (1.06 mM), ferrous sulphate (0.11 mM) and catalase (bovine liver, 1800 sigma units) in tris-HCl buffer (50 mM, p 7.5; tris stands for 2-amino-2-hydroxymethylpropane 1,3-diol) to a total volume of 1 ml, the incubation being carried out at 27° C.
• freeze dried material has the following the n.m.r. spectrum, the assignment of the various signals to protons in the structure (IV) shown hereinbefore being as follows:
• Mycelia (96 hours old/about 10 hours from the beginning of stationary phase) are harvested by filtration through a double-layer of cheese cloth.
• the cells collected are resuspended in 3 ⁇ (v/w) distilled water and suction-dried through Whatman filter paper (No. 52) in a Bucher funnel.
• the suction-dried mycelia are suspended in approximately 3 ⁇ (v/w) of 50 mM Tris-HCl buffer, pH 8.0, and disrupted in a Dyno Mill (Willy A Bacofen AG Maschinenfabrik, Basel) with 500 ml of 0.25 mm glass beads, being stirred at 3000 r.p.m. in the 600 ml glass head.
• protamine sulfate solution 6% w/v in distilled water
• the resulting solution is stirred for 20 minutes and centrifuged at 10,000 g for 30 minutes to remove precipitate.
• the supernatant collected above is brought to 55% saturation with ammonium sulphate (Sigma Grade I) and the precipitate is removed by centrifugation at 10,000 g for 30 minutes. The supernatant is then brought to 85% saturation with ammonium sulphate and enzyme protein is recovered as a precipitate after centrifugation at 10,000 g for 45 minutes.
• the precipitate obtained after 55-85% ammonium sulphate fractionation is dissolved in a minimum amount of Tris-HCl buffer (50 mM, pH 7.5, 0.015% w/v sodium azide), the final protein concentration being about 40 mg/ml. 80 ml of this protein solution is loaded onto a Sephadex G-75 (Pharmacia, coarse) column (3.7 cm ⁇ 110 cm) which has been pre-equilibrated with the same Tris-HCl buffer. The fractions with isopenicillin N synthetase activity are pooled and enzyme protein is concentrated by precipitation with 85% w/v ammonium sulphate followed by centrifugation at 10,000 g for 45 minutes.
• Tris-HCl buffer 50 mM, pH 7.5, 0.015% w/v sodium azide
• a highly purified enzyme preparation is obtained through ion-exchange chromatography of the enzyme fractions from Sephadex G75 column chromatography.
• the active fractions are pooled and loaded directly onto a DEAE-Sepharose CL-6B column (5 cm ⁇ 11.5 cm) which has been pre-equilibrated with Tris-HCl buffer (50 mM, pH 7.5, 0.015% w/v sodium azide).
• Tris-HCl buffer 50 mM, pH 7.5, 0.015% w/v sodium azide.
• the column is washed with about 500 ml of 50 mM NaCl in the same Tris-HCl buffer to remove unbound material.
• the column is then subjected to a gradient elution of NaCl in buffer (50 mM ⁇ 250 mM, total volume 800 ml, linear gradient), enzyme activity being eluted at about 100 mM NaCl.
• the preparation of the enzyme as obtained from the column is suitable for direct use.
• the activity of an amount of the enzyme may be quantitatively expressed in units of isopenicillin N synthetase activity, one each unit being defined as that amount of enzyme activity required to form 1 ⁇ mole of isopenicillin N per minute at 27° C. in a standard assay procedure employing incubation conditions similar to those described above.
• Example 2 ⁇ -(L- ⁇ -Aminoadipyl)-L-cysteinyl-(D-serine-O-methyl ether), prepared as described in Example 2, is incubated with purified isopenicillin N synthetase obtained as described under Example 3, the incubation medium being constituted as follows with the various component solutions being prepared in water.
• the final volume is adjusted to 5 ml with pH 7.5 tris buffer.
• a control is run in which 100 ⁇ l of water replaces the peptide solution.
• the incubation is carried out at 30° C. in a shaker (250 r.O.m) with exposure to the air for 30 minutes.
• the protein is precipitated from the mixture by the addition of acetone to a concentration of 70% by volume of acetone, then separated by centrifugation (5,000 r.p.m. for 10 minutes) and the supernatant freeze dried after removal of the acetone in vacuo to give the title compound which is believed to be a mixture of the 2R and 2S-isomers.
• the freeze dried product obtained from 210 ⁇ g of tripeptide as described in Example 3 was dissolved in pH 7.5 tris buffer to give a solution with a total volume of 1 ml. 100 ⁇ l of this solution was used for hole-plate inhibition assay for various as micro-organisms as listed in Table 1, the antibiotic activity of the penicillin against these micro-organisms being clearly illustrated by the results given in the table.
• the freeze dried product obtained from 500 ⁇ g of tripeptide as described in Example 4 was redissolved in 200 ⁇ l of water and 100 ⁇ l of this solution was incubated by hole-poate assay against S. aureus (both with and without the addition of ⁇ -lactamase I from Bacillus cereus 569/H/9. The remaining 100 ⁇ l of solution was diluted with another 100 ⁇ l of water and 100 ⁇ l of this more dilute solution was also assayed against S. aureus. This procedure was repeated to give successive levels of dilution. A control was used which contained none of the freeze dried solid. The results obtained are shown in the following Table 2 from which it will be seen that the antibiotic activity of the compound, which is clearly expressed in the absence of the ⁇ -lactamase, is destroyed by the action of this enzyme.

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Peptides Or Proteins (AREA)

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• 1984
  • 1984-08-22 GB GB848421279A patent/GB8421279D0/en active Pending
• 1985
  • 1985-08-20 GB GB08520775A patent/GB2163745B/en not_active Expired
  • 1985-08-20 EP EP85305911A patent/EP0174129A1/en not_active Withdrawn
  • 1985-08-21 DK DK379885A patent/DK379885A/da not_active Application Discontinuation
  • 1985-08-22 JP JP60185624A patent/JPS6169792A/ja active Pending
  • 1985-08-22 US US06/766,429 patent/US4722924A/en not_active Expired - Fee Related
  • 1985-08-22 FI FI853226A patent/FI853226A7/fi not_active Application Discontinuation

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